StarLighter Blood Simple Lysis Direct Amplification Probe qPCR Kit

Product Introduction

The StarLighter Probe qPCR Kit for Blood is a simple lysis kit for blood samples, enabling ideal qPCR amplification. Easy to use, the kit delivers results consistent with those obtained from traditionally purified nucleic acid samples. It includes blood sample lysis buffer and a 2× qPCR master mix.

This product's blood lysate contains a cell membrane lysing agent, protein denaturant, and nuclease inhibitor. To use, simply mix the lysate with blood in equal proportions, heat, centrifuge, and use the supernatant as a template. Combined with a specially developed blood-tolerant buffer system, this product inhibits nonspecific amplification while enabling specific and highly sensitive amplification for different template GC content and primer/probe Tm values. This product can be used for testing fresh EDTA-anticoagulated blood, stored at 4°C, or frozen at -20°C. It is suitable for SNP genotyping, qPCR probe quantification, and detection.

Product components

Note: Please store at the recommended storage temperature and avoid repeated freezing and thawing.

Storage and transportation conditions

Store at -25 ~ -15℃, transport at < 0℃.

Product Application

Nucleic acid release from blood samples;

SNP and allele typing;

Quantitative detection using qPCR probe method.

Precautions

1. Always ensure the product is completely melted and mixed thoroughly before use.
2. For convenience, StarLighter Blood DNA Release Buffer can also be stored at 2-8°C.
3. Use appropriate primer design software to design primers with a melting temperature around 60°C to better utilize the two-step cycling procedure.
4. Select the appropriate ROX according to different instruments.

Comparison table of instruments and ROX reference dyes:

experiment process

1. Blood sample processing
   • Transfer 75 μL of blood to a 5 mL centrifuge tube and add 75 μL of StarLighter Blood DNA Release Buffer (if crystals have precipitated before use, equilibrate at room temperature or heat to dissolve). Vortex to mix and centrifuge briefly. Then, heat the centrifuge tube in a metal bath at 95°C for 5 min.
   • After heating, remove the centrifuge tube and centrifuge at 12,000 rpm for 2 minutes (or at 4,000 rpm for 10 minutes). Remove the supernatant and use it as a template. Be careful not to aspirate the sediment on the wall or bottom of the centrifuge tube when removing the supernatant.

Note Generally, the volume of the supernatant after centrifugation is approximately 70% to 100% of the volume of blood added. It is recommended that the template added in the subsequent qPCR reaction account for 10% to 20% of the total reaction system. Amplification must be performed with the 2× StarLighter Probe qPCR Mix For Blood.

2. Amplification reaction system preparation
   • Before preparation, ensure that all components are thoroughly mixed;
   • Prepare the reaction system according to the following table:

1. The reaction system can be adjusted in the range of 5 to 25 μL (each component changes proportionally), and volumes >50 μL are not recommended.
2. The final concentrations of primers and probes in the reaction can be adjusted between 1 and 1 μM.
3. When using, it is best to prepare the common components into a premixed solution.
4. ROX is used according to the model.
   • Close the tube cap and centrifuge briefly.
5. Reaction procedure

qPCR standard procedure

1. The pre-denaturation time is set between 30 sec and 5 min. If the GC content of the template is high, the pre-denaturation time can be appropriately extended to 5 min.
2. Denaturation time: 10 sec for the standard program and 3 to 10 sec for the fast program.
3. The annealing/extension temperature needs to be determined based on the Tm values of the primers and probes, generally 50 ~ 65°C. For the first time, 60°C can be used for preliminary experiments.
4. The annealing/extension time can be adjusted between 20 and 45 seconds depending on the length of the amplified fragment.

Common Problems and Solutions

1. What are the causes and solutions for abnormal amplification curves?

Answer: ① If the amplification curve is not smooth, you need to recalibrate the system, increase the template concentration, and check whether ROX is used incorrectly.

②If the amplification curve breaks or declines, it may be caused by too high template concentration or inappropriate baseline selection range. You can reduce the template or reset the baseline.

③If the individual amplification curve drops suddenly, there may be bubbles in the reaction tube, so it should be centrifuged before loading.

2. What should I do if the reaction ends with no amplification curve?

Answer: ① It may be that the number of cycles is not enough: 40 cycles are generally set, but too many cycles will increase the background signal and reduce the credibility of the data.

② Confirm whether signal acquisition is set in the program: the two-step method generally collects signals during the annealing/extension stage; the three-step method collects signals during the extension stage.

③ Primer degradation: PAGE detection excludes the possibility of its degradation.

④The template concentration is too low: reduce the dilution and repeat the experiment, starting with a high concentration.

⑤ Template degradation: re-prepare the template.

3. Ct What if the value is too high?

Answer: ① Low amplification efficiency: Optimize reaction conditions, try three-step amplification or redesign synthetic primers.

② Low template concentration: Reduce the template dilution and start with high concentration.

③ Template degradation: re-prepare the template.

④The PCR product is too long: The recommended PCR product length is 80 to 150 bp.

⑤ PCR inhibitors are present: If the template is involved, increase the template dilution multiple or prepare the template again.

4. What should I do if the negative control shows obvious amplification?

Answer: ① Contamination of the reaction system: Repeat the experiment with new reagents, water, primers, etc.; operate in a clean bench to reduce aerosol contamination.

② Improper primer design: Redesign and synthesize primers.

5. What should I do if the standard curve has poor linearity?

Answer: ① Sample addition error: increase the template dilution multiple and increase the sample addition volume.

②Degradation of standard sample: re-prepare the standard sample.

③The template concentration is too high: increase the template dilution multiple.

6. What to do if the experiment has poor reproducibility?

Answer: ① Inconsistent sample volume: Use a better pipette; dilute the template by a high multiple and increase the sample volume appropriately.

②The template concentration is too low: The lower the template concentration, the worse the repeatability. Reduce the template dilution or increase the sample volume.